The increasing application of recombinant DNA technology to engineer novel microorganism which are industrially useful have caused concerns in the general public over the potential risks involved. These concerns are primarily related to the potential harm to humans and to undesirable and/or uncontrollable ecological consequences upon deliberate or unintentional release of such genetically engineered microorganisms (GEMs) into the environment. These concerns have led to the establishment of official guidelines for the safe handling of GEMs in laboratories and production facilities where such organisms are applied. Up till now, such guidelines have primarily been directed to measures of physically containing GEMs in laboratories and production facilities with the aim of reducing the likelihood that workers in such facilities were contaminated, or that the GEMs were to escape from their primary physical environment, such as a fermentation vessel.
It is presently being recognized that the level of safety in the handling of GEMs can be increased by combining physical containment measures with biological containment measures to reduce the possibility of the survival of the genetically engineered organisms if they were to escape from their primary environment.
Lately, however, concerns have become increasingly focused on potential risks related to deliberate release of GEMs to the outer environment and to the use of GEMs as live vaccines. In this connection there is a strongly felt need to have biological containment systems which subsequent to the environmental release of the GEMs or their administration as vaccines to a human or an animal body, effectively kill the released organisms in a controlled manner or which limit the function of the released GEMs to an extent where such GEMs are placed at a significant competitive disadvantage whereby they will eventually be ousted by the natural microflora of the environment to which they are released.
The first systems of biological containment were based on the use of “safe” cloning vectors and debilitated host bacteria. As examples, it has been suggested to select vectors which lack transfer functions or which naturally have a very narrow host range. Examples of debilitated host bacteria are E. coli mutants having an obligate requirement for exogenous nutrients not present or present in low concentrations outside the primary environment of the GEMs.
Other suggested biological containment systems have been based on mechanisms whereby the vector is restricted to the GEMs e.g. by using a plasmid vector with a nonsense mutation in a gene, the expression of which is indispensable for plasmid replication or a suppressor mutation in the chromosome, said mutation blocking translational read-through of the message of the gene. A further approach is to maintain the rDNA stably in the host by integrating it into the chromosomes of the GEMs.
Recently, an alternative biological containment strategy has been developed in which a recombinant vector is endowed with a gene encoding a cell killing function which gene is under the control of a promoter only being expressed under certain environmental conditions, such as conditions prevailing in an environment outside the primary environment of the GEMs, or when the vector is unintentionally transferred to a secondary host, or the expression of which is stochastically induced. By using incorporation in a GEM of such a cell killing function and selecting appropriate regulatory sequences, vectors can be constructed which are contained in the primary host cell and/or in a primary physical environment. A cell killing function as defined herein may also be referred to as an active biological containment factor.
If a stochastically induced mechanism of expression regulation is selected for such a biological containment system, a population of GEMs containing the system will, upon release to the outer environment or if used as a live vaccine, be subjected to a random cell killing which will lead to an increase of the doubling time of the host cell population or eventually to the disappearance of the organisms.
The above-mentioned genes encoding cell killing functions are also frequently referred to as “suicide” genes, and biological containment systems based upon the use of such genes, the expression of which are regulated as defined above, are commonly described as conditional lethal systems or “suicide” systems. Up till now, several such cell killing functions have been found in bacterial chromosomes and in prokaryotic plasmids. Examples of chromosomal genes having cell killing functions are the gef (Poulsen et al., 1990) and relF (Bech et al., 1985) genes from E. coliK-12. Examples of plasmid encoded suicide genes are hok and flmA (Gerdes et al., 1986) genes isolated from plasmids R1 and F, respectively, the snrb gene also isolated from plasmid F (Akimoto et al., 1986) and the pnd gene isolated from plasmids R16 and R483 (Sakikawa et al., 1989 and Ono et al., 1987). Common features of these genes are that they are transcribed constitutively, regulated at a post-transcriptional level, and that they all encode small toxic proteins of about 50 amino acids and that their translation is controlled by antisense RNA. The application of the hok gene in a biological containment system has been disclosed in WO 87/05932.
An alternative biological containment system is disclosed in WO 95/10614 which is based on the use of genes, the expression of which in a cell where the gene is inserted, results in the formation of mature forms of exoenzymes which are hydrolytically active in the cytoplasm of the cell and which can not be transported over the cell membrane. When such enzymes are expressed, the normal function of the cell becomes limited to an extent whereby the competitiveness, and hence the survival, of a population of such cells is reduced.
The stable maintenance of low copy-number plasmids in bacteria is secured by a number of plasmid-borne gene systems, one of which is based on killing of plasmid-free cells (also termed post-segregational killing). This regulated killing is based on a toxin-antidote principle, i.e. a two-component system comprising a stable toxin and an unstable antidote for the toxin. One such system, which is referred to as a proteic killer gene system is based on protein toxins and protein antidotes (reviewed by Jensen and Gerdes, 1995). The natural function of such systems is to provide stable maintenance of plasmids and it has not been suggested previously to utilize the systems as the basis for confining GEMs to a particular environment.
The E. coli relB operon encodes three genes, relB, relE and relF (Bech et al., 1985). It has now been found that relE encodes a cytotoxin whose overproduction is lethal to host cells and that the relB gene encodes an antitoxin that prevents the lethal action of RelE. When present on a plasmid, the relBE operon was able to stabilize the inheritance of a mini-R1 test plasmid. It was also found that relBE homologous gene systems are found in a wide variety of Gram-negative and Gram-positive bacteria and in Archae.
These results show that the relBE genes constitute a new ubiquitously occurring family of gene systems that belongs to the proteic plasmid stabilization systems.
These findings has opened up for an alternative, highly effective and versatile biological containment system as it is described in the following. Importantly, it has been discovered that such a system involves the significant advantage that the frequency of spontaneously occurring mutants of microorganisms that have become resistant to the lethal effect of these cytotoxins is very low. This implies that this biological containment system is very safe.